【摘要】：背景皮膚上皮干細(xì)胞作為種子細(xì)胞用于人造皮膚的制造和傷口修復(fù)已有多年歷史,但通常所用的干細(xì)胞為單一分化潛能的表皮干細(xì)胞,無法重建任何皮膚附屬器官,實(shí)現(xiàn)皮膚的全部功能。研究表明毛囊干細(xì)胞具有修復(fù)表皮,分化為毛發(fā)、皮脂腺、汗腺的多分化潛能,是傷口修復(fù)和人造皮膚的理想種子細(xì)胞。然而人類正常皮膚組織中,毛囊干細(xì)胞的比例極低,一根毛囊僅能提取200-400個(gè)毛囊干細(xì)胞,而臨床治療所需的毛囊干細(xì)胞通常需要百萬數(shù)量級(jí)以上,原代毛囊干細(xì)胞數(shù)量遠(yuǎn)不能滿足需求。而毛囊干細(xì)胞傳統(tǒng)體外培養(yǎng)需要滋養(yǎng)層細(xì)胞及血清培養(yǎng),但這樣的細(xì)胞產(chǎn)品不能用于臨床治療,因此急需建立支持人類毛囊干細(xì)胞體外無血清無滋養(yǎng)層細(xì)胞的擴(kuò)增技術(shù)。運(yùn)用小分子抑制劑可以精確對(duì)信號(hào)通路進(jìn)行激活或抑制,在細(xì)胞培養(yǎng)研究領(lǐng)域已經(jīng)取得一些進(jìn)展。小分子抑制劑Y-27632為Rock的抑制劑,可以促使人胚胎干細(xì)胞、角質(zhì)上皮細(xì)胞增殖,抑制細(xì)胞的凋亡;A83-01可以抑制TGF-βI型受體ALK5的轉(zhuǎn)錄活性,協(xié)同CHIR-99021可將成熟肝細(xì)胞逆轉(zhuǎn)成干細(xì)胞,A83-01協(xié)同DMH-1、CHIR-99021、Y-27632在無血清無滋養(yǎng)層細(xì)胞的條件下能夠在體外大量擴(kuò)增某些上皮細(xì)胞。以上這些結(jié)果表明在細(xì)胞培養(yǎng)中利用小分子抑制劑可以一定程度上彌補(bǔ)無血清培養(yǎng)營養(yǎng)不足導(dǎo)致的細(xì)胞生物學(xué)性狀改變,開拓干細(xì)胞培養(yǎng)的新局面。目前,A83-01、DMH-1、CHIR-99021、Y-27632在毛囊干細(xì)胞體外無血清擴(kuò)增的作用尚未見相關(guān)報(bào)道。本課題擬建立符合本實(shí)驗(yàn)室及貼近臨床運(yùn)用的人毛囊干細(xì)胞采集純化保存的規(guī)范化流程,在此基礎(chǔ)上研究小分子抑制劑A83-01、DMH-1、CHIR-99021、Y-27632對(duì)人毛囊干細(xì)胞體外無血清擴(kuò)增的影響,為人毛囊干細(xì)胞臨床應(yīng)用及研究提供一定的理論基礎(chǔ)和技術(shù)支持。目的1.建立人毛囊干細(xì)胞庫及細(xì)胞采集純化保存的規(guī)范化流程,并行鑒定;2.探索體外無血清培養(yǎng)人毛囊干細(xì)胞方法,優(yōu)化擴(kuò)增人毛囊干細(xì)胞及維持干性的條件。方法1.人毛囊外根鞘細(xì)胞獲取:采用Dispase II初步消化帶毛囊組織,顯微鏡下拔取毛囊,剔除皮脂腺,離斷毛乳頭,0.05%Trypsin-EDTA消化獲得人毛囊外根鞘細(xì)胞單細(xì)胞懸液。免疫熒光染色觀察處理前后干細(xì)胞變化。2.差速貼壁富集毛囊干細(xì)胞及原代細(xì)胞培養(yǎng):利用含人I型膠原的COATING MATRIX KIT進(jìn)行包被培養(yǎng)皿,15min差速貼壁分離毛囊干細(xì)胞,利用CNT-PR原代上皮培養(yǎng)液添加Y-27632進(jìn)行原代細(xì)胞培養(yǎng)。流式分析及免疫熒光觀察純化結(jié)果。3.人毛囊干細(xì)胞的培養(yǎng)條件探索:分組培養(yǎng)毛囊干細(xì)胞,對(duì)照組培養(yǎng)基(CNT組)、實(shí)驗(yàn)組1(A83-01組)、實(shí)驗(yàn)組2(DMH-1組)、實(shí)驗(yàn)組3(A83-01+DMH-1組)、實(shí)驗(yàn)組4(CHIR-99021)、實(shí)驗(yàn)組5(A83-01+DMH-1+CHIR-99021組),CCK-8行細(xì)胞分組條件下短期培養(yǎng)細(xì)胞增殖速度,傳代培養(yǎng)時(shí),每代記錄接種細(xì)胞及收細(xì)胞數(shù)目,免疫熒光染色K15細(xì)胞比例,計(jì)算細(xì)胞擴(kuò)增總量及K15陽性細(xì)胞擴(kuò)增情況。結(jié)果1、免疫熒光染色定位毛囊干細(xì)胞于毛囊的中段,顯微外科除去毛乳頭及皮脂腺,初步提高了毛囊干細(xì)胞純化效果;2、以含人I型膠原的COATING MATRIX KIT進(jìn)行差速貼壁分離毛囊干細(xì)胞,有效富集CD200+/CD49+毛囊干細(xì)胞(P0.05),同時(shí)避免傳統(tǒng)利用小鼠IV型膠原差速貼壁帶來的異種蛋白侵入危險(xiǎn);3、成功在無血清、無滋養(yǎng)層、培養(yǎng)基成分限定的條件下進(jìn)行原代干細(xì)胞體外培養(yǎng)16±3天,每根毛囊可取得4.5±0.7×104 K15陽性細(xì)胞;4、添加A83-01、Y-27632或者添加A83-01、DMH-1、Y-27632于CNT-PR培養(yǎng)基較單純添加Y-27632于CNT-PR培養(yǎng)基或其他分組,能提高總體細(xì)胞及K15陽性細(xì)胞增殖速度,縮短擴(kuò)增時(shí)間(P0.05);5、本實(shí)驗(yàn)流程可在無血清、無滋養(yǎng)層、培養(yǎng)基成分限定的條件下進(jìn)行毛囊干細(xì)胞的培養(yǎng)擴(kuò)增,利于實(shí)驗(yàn)研究的分析和臨床應(yīng)用。結(jié)論:我們?cè)诒苊猱惙N蛋白或基因干擾的條件下,利用差速貼壁法純化毛囊干細(xì)胞,在體外無血清擴(kuò)增,6周內(nèi)1根毛囊擴(kuò)增出的毛囊干細(xì)胞可達(dá)百萬數(shù)量級(jí),貼近臨床運(yùn)用,并發(fā)現(xiàn)A83-01及DMH-1可以提高毛囊干細(xì)胞擴(kuò)增效率及干性維持。
[Abstract]:Background skin epithelial stem cells have been used as seed cells for the manufacture and repair of artificial skin for many years. However, the stem cells used as a single differentiation potential stem cells can not reconstruct any skin accessory organs to achieve all the functions of the skin. The multiple differentiation potential of sebaceous glands and sweat glands is the ideal seed cell for wound repair and artificial skin. However, in human normal skin tissue, the proportion of hair follicle stem cells is very low. One hair follicle can only extract 200-400 hair follicle stem cells, and the hair follicle stem cells needed for clinical treatment usually require more than a million orders of grade, original hair follicle stem cells. The traditional culture of hair follicle stem cells in vitro requires the culture of trophoblast cells and serum, but this kind of cell product can not be used for clinical treatment. Therefore, it is urgent to establish the amplification technology that supports human hair follicle stem cells without serum-free trophoblast cells in vitro. In the field of cell culture, some progress has been made in the field of cell culture. Small molecular inhibitor Y-27632 is an inhibitor of Rock, which can promote the proliferation of human embryonic stem cells, keratinocytes and inhibit cell apoptosis; A83-01 can inhibit the transcriptional activity of TGF- beta I receptor ALK5 and reverse the maturation of mature hepatocytes into dry cells with CHIR-99021. Cells, A83-01 and DMH-1, CHIR-99021, and Y-27632 can amplify a large number of epithelial cells in vitro under the conditions of serum-free and non trophoblastic cells. These results suggest that the use of small molecular inhibitors in cell culture can partly compensate for the changes in cell biological properties caused by no serum-free culture, and to open up dry cells. At present, the role of A83-01, DMH-1, CHIR-99021 and Y-27632 in the serum-free expansion of hair follicle stem cells in vitro has not been reported. This topic intends to establish a standardized process for the collection, purification and preservation of human hair follicle stem cells, which is in accordance with this laboratory and close to clinical application. On this basis, the small molecule inhibitors, A83-01, DMH-1, are studied. The effect of CHIR-99021 and Y-27632 on the serum-free expansion of human hair follicle stem cells in vitro provides a theoretical basis and technical support for the clinical application and research of human hair follicle stem cells. Objective 1. to establish a standardized process for human hair follicle stem cell bank and cell collection and purification. 2. to explore the serum-free culture of human hair follicle stem cells in vitro Methods to optimize the expansion of human hair follicle stem cells and maintain the dry condition. Methods 1. human hair follicle outer root sheath cells were obtained: using Dispase II preliminary digestive tract hair follicle tissue, extracting hair follicles, removing sebaceous glands, breaking off hair nipples, and digesting human hair follicle outer root sheath cells single cell suspension. Immunofluorescence staining observation place. Pre and post stem cell changes.2. differential adherent enrichment of hair follicle stem cells and primary cell culture: using COATING MATRIX KIT containing human I collagen to pack culture dish, 15min differential adherent separation of hair follicle stem cells, primary cultured cell culture with Y-27632 in CNT-PR primary culture medium. Flow analysis and immunofluorescence observation and purification Fruit culture conditions of.3. human hair follicle stem cells: group culture hair follicle stem cells, control group (group CNT), experimental group 1 (group A83-01), experimental group 2 (DMH-1 group), experimental group 3 (A83-01+DMH-1 group), experimental group 4 (CHIR-99021), experimental group 5 (A83-01+DMH-1+ CHIR-99021 group), CCK-8 row cell group conditions for short-term culture of cell proliferation speed, transmission, transmission of cells. At the time of culture, the inoculation cells and the number of cells collected, the proportion of immunofluorescent staining K15 cells, the total amount of cell amplification and the amplification of K15 positive cells were calculated. Results 1, immunofluorescence staining was used to locate the hair follicle stem cells in the middle part of the hair follicle, microsurgical removal of dermal papilla and skin fat gland, initially raising the purification effect of hair follicle stem cells; 2, COATING MATRIX KIT containing human type I collagen was used for differential adhesion separation of hair follicle stem cells, effectively enriching CD200+/CD49+ hair follicle stem cells (P0.05), and avoiding the invasion of xenogeneic proteins caused by the differential adhesion of IV collagen in mice. 3, the primary stem cells were successfully carried out under the condition of serum-free, no trophoblastic layer and limited culture medium. For 16 + 3 days in vitro culture, 4.5 + 0.7 x 104 K15 positive cells could be obtained in each hair follicle. 4, adding A83-01, Y-27632 or adding A83-01, DMH-1, Y-27632 on CNT-PR culture medium could increase the proliferation rate and shorten the amplification time (P0.05) of the whole cell and K15 positive cells (P0.05); 5. It is beneficial to the analysis and clinical application of hair follicle stem cells in the condition of no serum-free, no trophoblast and culture medium. Conclusion: we have purified hair follicle stem cells with differential adherent method under the condition of avoiding heterogenous protein or gene interference, and expanded the hair follicle without serum in vitro, and amplified 1 hair follicles within 6 weeks. Hair follicle stem cells can reach one million orders of magnitude, which is close to clinical application. It is found that A83-01 and DMH-1 can enhance the amplification efficiency and dry maintenance of hair follicle stem cells.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R622

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